Uses of Yerba Santa

ABSTRACT

Methods of in vitro propagation of plants of the genus  Eriodictyon  are described, including, in particular embodiments, plants of the species  E. californicum, E. trichocalyx  and  E. sessilifolium . Methods of producing transgenic plants of the genus  Eriodictyon  are also described, along with methods of producing recombinant proteins in such plants. Compositions and methods for administering recombinant proteins produced in these plants are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Non-Provisional applicationSer. No. 12/487,942 filed on Jun. 19, 2009, which claims priority fromU.S. Provisional Application No. 61/074,376 filed on Jun. 20, 2008, thedisclosures of which are herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe United States Department of Agriculture, Grant Number SCA58-1275-4-303. The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

The medicinal plant Yerba Santa, member of the Waterleaf Family, genusEriodictyon, has a long tradition of use. Yerba Santa (includingEriodictyon californicum, Eriodictyon trichocalyx and other relatedspecies) has been used in treating respiratory conditions, includingcolds, cough, asthma and bronchitis. This herb has also been foundeffective for a number of other symptoms including gastrointestinaldisorders, fatigue, rheumatism, and allergies. Biochemical analyses haveconfirmed Yerba Santa to have flavonoids that show promise asanti-carcinogens (Liu Y L, Ho D K, Cassady J M, Cook V M, Baird W M(1992) J Nat Prod 55:357-363).

For medical purposes, Yerba Santa has been used as either a dry herb oran extract. (Heizer R F, Elsasser A B (1980) The natural world of theCalifornia Indians, University of California Press, Berkeley & LosAngeles, Calif. 271 pp.). Many herbal stores carry different productscontaining Yerba Santa such as, for example, leaf powder, extracts, leaftea, and cream. The fluid Yerba Santa extracts have been used in food,beverages, pharmaceuticals and cosmetics.

Yerba Santa is a perennial evergreen shrub (1-2 meters) that grows indry, hilly areas of California and Northern Mexico. During dry months,the leaves become hard and resinous in order to hold and conserve water.When applied to mucosal surfaces, the herb preparation holds the aqueouscomponent in contact with cells, reestablishing mucopolysaccharides. Itis hypothesized that this property may facilitate the adherence to themucosa of compositions such as, for example, pharmaceutical agents.

In recent years, there has been an increased interest in massmultiplication of this unique plant. In vitro culture techniques havebeen used successfully for large scale production of many medicinallyimportant plant species. However, methods for in vitro propagation andtissue culture techniques for Yerba Santa have not previously beendescribed.

Plant biotechnology has provided successful tissue culture andtransformation technologies for a variety of plants. However, the use ofbiotechnological tools in medicinal plant science has been very limitedas compared to other crops. Nevertheless, in recent years suchtechniques have been developed for number of important medicinal plantssuch as Ginkgo biloba, Digitalis lanata, Artenisia annua, Papaversomniferum, Camptotheca acuminate, Ophiorrhiza prostrate, and Menthapiperita. Although Yerba Santa is a very important medicinal plant,there have been no techniques described for in vitro propagation, cellculture cultivation, regeneration and transformation of this perennialshrub.

Modern plant biotechnology has opened new avenues for producingrecombinant molecules, including, but not limited to, vaccines (HanssonM, Nygren P A & Stahl S (2000) Biotechnol Appl Biochem 32:95-107;Daniell H, Streatfield S J & Wycoff K (2001) Trends Plant Sci 6:219-226;Ma J K C, Drake P M W & Christou P (2003) Nat Rev Genet. 4:794-805;Koprowski H (2005) Vaccine 23:1757-1763; Pogrebnyak N., Golovkin M.,Andrianov V., Spitsin S., Smirnov Y., Egolf R., Koprowski H (2005) ProcNatl Acad Sci USA 102: 9062-9067; and Golovkin M., Spitsin S., AndrianovV., Smirnov Y., Xiao Y., Pogrebnyak N., Markley K., Brodzik R., GlebaY., Isaacs S N, Koprowski H. Proc Natl Acad Sci USA (2007), 104:6864-6869; Goldstein D A, Thomas J A (2004) QJ Med 97:705-716) ormicrobicides (O'Keefe B. et al. Proc Natl Acad Sci USA (2009), 106:6099-6104). This approach has become an attractive alternative to othertechnologies since it is associated with low production cost, overallsafety, and scalability potential. A potential benefit of using plantsfor vaccine production is the possibility of applying preparationsdirectly to bodily surfaces such as, for example, mucosal surfaces(Goldstein D A, Thomas J A (2004) QJ Med 97:705-716; Giddings G, AllisonG, Brooks D, Carter A (2005) Nat Biotechnol 18:1151-1155; Pogrebnyak N.Markley K., Smirnov Y., Brodzik R., Bandurska K., Koprowski H., GolovkinM (2006) Plant Sci. 171: 677-685); Golovkin M., Spitsin S., AndrianovV., Smirnov Y., Xiao Y., Pogrebnyak N., Markley K., Brodzik R., GlebaY., Isaacs S N, Koprowski H. Proc Natl Acad Sci USA (2007), 104:6864-6869; O'Keefe B. et al. Proc Natl Acad Sci USA (2009), 106:6099-6104).

BRIEF SUMMARY OF INVENTION

The invention relates to a transgenic plant of the genus Eriodictyon, inparticular plants of the species E. californicum, E. trichocalyx or E.sessilifolium. In one aspect, the transgenic plant expresses arecombinant protein selected from the group consisting of antigens,microbicides, antibodies, hormones, enzymes, blood components,interferons, and anticoagulants. In a particular embodiment, the antigenis a viral protein such as an avian influenza HA1 antigen. In anotherembodiment, the microbicide is an antiretroviral such as griffithsin.

In another embodiment, the present invention relates to a method fortransforming a plant tissue, particularly plant tissue from a plantspecies of the genus Eriodictyon including the steps of: inoculating atransformable plant tissue with an Agrobacterium suspension, theAgrobacterium containing at least one genetic component encoding adesired protein capable of being transferred to the transformable planttissue and of directing the expression of the desired protein in theplant tissue; co-cultivating the plant tissue with the Agrobacterium;transferring the plant tissue to recovery media containing an antibioticfor eliminating the Agrobacterium; and selecting transformed planttissue.

The invention also relates to a method for producing a recombinantprotein in a transgenic plant of the genus Eriodictyon including thesteps of: providing a transgenic plant that has been regenerated from atransformed plant cell or tissue of the genus Eriodictyon and thatexpresses a recombinant protein; and recovering the protein expressed inthe transgenic plant.

One embodiment of the present invention relates to a method ofdelivering a recombinant protein to a subject including providingharvested material from a transgenic plant of the genus Eriodictyon thatexpresses a recombinant protein; and administering the harvestedmaterial to the subject in an amount necessary to deliver an effectiveamount of the recombinant protein. In a particular aspect, therecombinant protein is an antigen and the harvested material isadministered in an amount sufficient to induce an immune response in thesubject. In another aspect, the recombinant protein is a microbicide andthe harvested material is administered in an amount sufficient toprovide a prophylactic effect.

In another embodiment, the invention relates to a method of propagatingin vitro a plant of the genus Eriodictyon, the method including thesteps of: excising a stem segment of the plant; and incubating thesegment in a growth medium comprising a cytokinin; whereby the segmentproduces a shoot. In another aspect, the method further includesexcising the shoot; and incubating the excised shoot in mediumcomprising an auxin, whereby the shoot produces a root.

In another embodiment, the method of propagating in vitro a plant of thegenus Eriodictyon further includes incubating the shoot for at leastthree weeks, whereby the shoot produces a leaf; cutting a segment fromthe leaf; placing the segment in a culture medium comprising one or moreof benzylaminopurine, naphthaleneacetic acid or2,4-dichlorophenoxyacetic acid; and incubating the segment in the dark,whereby the segment develops callus tissue.

In another aspect, the invention relates to a method of producing a cellsuspension culture of a plant of the genus Eriodictyon including thesteps of: excising a portion of the callus tissue produced according tocertain aspects of the invention;

placing the callus tissue in a liquid medium comprising2,4-dichlorophenoxyacetic acid to form a cell suspension; and incubatingthe cell suspension in the dark while agitating the medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates in vitro propagation of Yerba Santa. (A) Yerba SantaE. trichocalyx stem segments, 3 days on MS medium with 1 mg/l zeatin (B)E. trichocalyx stem segments produced multiple shoots after 5 weeks onMS medium with 1 mg/l zeatin. (C) Root formation in E. sessilifoliumafter 10 days on MS medium. (D) Root induction in E. trichocalyx on MSmedium with 1 mg/l IBA, view of bottom of plastic culture box. (E) Rootinduction in E. californicum on MS medium with 1 mg/l IBA. (F) Growthand development of E. trichocalyx plant in vitro. (G) E. californicumplant in a pot, 2 weeks after transfer. (H) E. trichocalyx plant in apot, 4 weeks after transfer.

FIG. 2 illustrates Yerba Santa callus and cell suspension cultures. (A)Callus propagation on MS medium with 1 mg/l 2.4-D (E. trichocalyx). (B)Callus propagation on MS medium with 1 mg/l 2,4-D (E. californicum). (C)Cell suspension (E. trichocalyx), 1 day in liquid MS medium with 0.5mg/l 2,4-D. (D) Cell suspension (E. trichocalyx) after 2 weeks in liquidMS medium with 0.5 mg/l 2,4-D.

FIG. 3 is a graph illustrating the growth rate of a Yerba Santa E.trichocalyx cell suspension.

FIG. 4 is a graph illustrating shoot regeneration efficiency in threeYerba Santa species. Regeneration efficiency is expressed as the percentof explants producing shoots on MSR medium during 6 weeks.

FIG. 5 illustrates an assessment of regeneration and transformationprocedures for Yerba Santa (E. trichocalyx) (A) Regeneration throughcallus formation; (B) Direct regeneration from leaf explant on MSRmedium; (C) Transgenic plant expressing Avian flu antigen on MST-5medium containing 100 mg/l kanamycin; (D) Multiplication of transgenicline on MSP medium.

FIG. 6 illustrates a transgenic Yerba Santa (E. trichocalyx) expressingAvian flu antigen. (A) Transgenic plant on root induction medium; (B)Transgenic plant in a pot.

FIG. 7 is a Western blot analysis of transgenic Yerba Santa plantsshowing expression of recombinant HA1 antigen. (A) SDS/PAGE; (B) Westernblot using HA1 antigen-specific antibodies; (C) Western blos using c-Mycantibodies.

DETAILED DESCRIPTION OF THE INVENTION

Plants have emerged as modern efficient systems for production anddelivery of recombinant products, including, but not limited to,microbicides and antigens. When applied on a mucosal surface,preparations of this herb have a capacity to hold aqueous components incontact with mucosal cells that may facilitate adherence ofpharmaceuticals to the mucosa. Accordingly, Yerba Santa may be useful asa vehicle for effective delivery of recombinant molecules, such asrecombinant microbicides or vaccines, to body surfaces including mucosalsurfaces, including, for example, intranasal and oral surfaces. Abenefit of using plants for production of recombinant drugs is that theyallow direct mucosal (and thus needle-free) administration of thepharmaceutical. For mucosal delivery of a vaccine antigen to a subject,the extended mucosal exposure to such a plant-based vaccine maysignificantly increase immune response in the subject. Microbicidesreduce the infectivity of microbes, such as viruses or bacteria, byaverting infection at the mucosal surfaces. Accordingly, a plant baseddelivery system that is directly applied to mucosal surfaces is ideallysuited for the administration of microbicides.

Yerba Santa is suitable for human consumption, has been used in medicinefor centuries, and is widely used in the food industry. According tocertain aspects of the invention, modern plant biotechnology techniqueshave been developed for this valuable medicinal plant, including thedevelopment of transgenic Yerba Santa plants. In certain embodiments, invitro propagation, regeneration and transformation systems are providedfor Yerba Santa species. In certain aspects, cell culture technologiesare provided for Yerba Santa that may offer economically favorablemethods for production of large amounts of recombinant pharmaceuticals.For example, cell suspensions may be used to produce large amounts ofbiopharmaceuticals in bioreactors under controlled conditions to achieveuniform, high quality products.

One aspect of the present invention is a transgenic plant; preferablythe plant is a member of the genus Eriodictyon. Preferably, the plant isE. californicum, E. trichocalyx or E. sessilifolium.

In certain embodiments, the invention relates to a recombinant proteinexpressed by a transgenic plant according to aspects of the invention.

As used herein, a “recombinant protein” means that the protein, whethercomprising a native or mutant primary amino acid sequence, is obtainedby expression of a gene carried by a recombinant DNA molecule in a cellother than the cell in which that gene and/or protein is naturallyfound. In other words, the gene is heterologous to the host in which itis expressed. This protein may include an entire (full-length) proteinor a polypeptide molecule, or may comprise a protein or polypeptidefragment suitable for a particular purpose. Preferably, the recombinantproteins expressed by the transgenic plant are suitable for use aspharmaceuticals, including, but not limited to, antigens, microbicides,antibodies, hormones, enzymes, blood components, including, but notlimited to, coagulation factors, interferons, and anticoagulants.

As used herein, “antigen” may include a single antigen or a plurality ofantigens as long as at least one antigen is included which, whenadministered in a sufficient amount, can induce an immune response in asubject. The term “antigen” also includes any portion of an antigen,e.g., the epitope, which can induce an immune response. Preferably, theone or more antigens will produce a sufficient immune response to conferresistance to infection upon the recipient of the antigen. Examples ofimmunogenic or antigenic molecules that may be useful include, withoutlimitation, viral antigens such as the entirety or portions of:Hepatitis virus B surface antigen, Malaria parasite antigen, Influenza AH1N1 antigen, Rabies virus glycoprotein, Escherichia coli heat-labileenterotoxin, Human rhinovirus 14 (HRV-14), human immunodeficiency virustype (HIV-1) epitopes, Norwalk virus capsid protein, Diabetes-associatedautoantigen, Mink Enteritis Virus epitope, Foot and mouth disease virusVP1 structural protein, Cholera toxin B subunit, Human insulin-Choleratoxin B subunit fusion protein, Human cytomegalovirus glycoprotein B, S.mutans, respiratory syncytial virus antigens (F1, F2, G), tetanus toxinfragment C, diphtheria toxin, S1 subunit of pertussis toxin and SARSS-glycoprotein.

In certain embodiments, the protein expressed by the transgenic plantcomprises an antigen protein, preferably a viral protein, morepreferably an avian influenza HA1 antigen.

As used herein, “microbicide” refers to any compound or substance whosepurpose is to reduce the infectivity of microbes, such as viruses orbacterial. Examples of microbicides that may be useful include, withoutlimitation, griffithsin, the fusion inhibitor C52, RANTES analoguePSC-RANTES, and lectin cyanovirin-N (CV-N).

In certain embodiments, the recombinant protein expressed by thetransgenic plant comprises a microbicide, preferably an antiretroviralmicrobicide, more preferably an HIV entry inhibitor, even morepreferably griffithsin. As used herein, “griffithsin”, which has beenshown to be a highly potent HIV entry inhibitor, refers to a121-amino-acid protein isolated from the red algae Griffithsia or activemutants or fragments thereof. Griffithsin. Mori T, O'Keefe B R, Sowder RC, et al. (2005), “Isolation and characterization of griffithsin, anovel HIV-inactivating protein, from the red alga Griffithsia sp”, J.Biol. Chem. 280 (10): 9345-53.

Examples of antibodies that may be useful include, without limitation,monoclonal antibodies (mAbs) and secretory IgA (sigA). Antibody formatsmay include, without limitation, full-size, Fab fragments, single-chainantibody fragments, bi-specific scFv fragments, membrane anchored scFv,chimeric antibodies and humanized antibodies.

Examples of hormones that may be useful include, but are not limited toinsulin, somatotropin, and erythropoietin.

Examples of enzymes that may be useful include, without limitation,α-1-antitrypsin, aprotinin, Antiotensin-1-converting enzyme, andGlucocerebrosidase

Examples of blood components that may be useful include, withoutlimitation, serum albumin, including human serum albumin, andhemoglobin.

Examples of interferons that may be useful include, without limitation,interferon-α.

Examples of anticoagulants that may be useful include, withoutlimitation, protein C (serum protease) and hirudin.

In certain embodiments, the invention relates to methods for producingrecombinant proteins in transgenic plants comprising the steps of:constructing a plasmid vector or a DNA fragment by operably linking aDNA molecule comprising a sequence encoding a protein to a promotercapable of directing the expression of the protein in the plant;transforming a plant cell with the plasmid vector or DNA fragment tocreate a transgenic plant cell; and recovering the protein expressed inthe plant cell for use. Preferably, the plant is a member of the genusEriodictyon. Preferably, the plant is E. californicum, E. trichocalyx,or E. sessilifolium. Preferably, the recombinant proteins are suitablefor use as pharmaceuticals, including, but not limited to, antigens;microbicides; antibodies; hormones; enzymes; blood components,including, but not limited to, coagulation factors; interferons, andanticoagulants.

Exogenous DNA constructs used for transforming plant cells will comprisethe coding sequence of recombinant protein desired to be expressed andusually other elements such as, but not limited to introns, 5′ and 3′untranslated regions, and promoters. In certain embodiments, theutilization of a strong promoter, such as the Rubisco promoter operablylinked to the coding sequence of the desired recombinant protein,provides large amounts of recombinant protein in Yerba Santa leaves.

As is well known in the art, DNA constructs for use in transformingplants and expressing a coding sequence typically also comprise otherregulatory elements in addition to a promoter, such as but not limitedto 3′ untranslated regions (such as polyadenylation sites), transit orsignal peptides and marker coding sequences elements.

During transformation, exogenous DNA may be introduced randomly, i.e. ata non-specific location, in the plant genome. In some cases, it may beuseful to target an exogenous DNA insertion in order to achievesite-specific integration, e.g. to replace an existing gene sequence orregion in the genome. In some other cases it may be useful to target anexogenous DNA integration into the genome at a predetermined site fromwhich it is known that gene expression occurs.

In practice DNA is introduced into only a small percentage of targetcells in any one experiment. Marker genes are used to provide anefficient system for identification of those cells that are stablytransformed by receiving and integrating an exogenous DNA construct intotheir genomes. Preferred marker genes provide selective markers whichconfer resistance to a selective agent, such as an antibiotic orherbicide. Potentially transformed cells are exposed to the selectiveagent. In the population of surviving cells will be those cells where,generally, the resistance-conferring coding sequence has been integratedand expressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.Useful selective marker genes include those conferring resistance toantibiotics such as kanamycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4) or resistance to herbicides such asglufosinate (bar or pat) and glyphosate (EPSPS; CP4).

Means for transforming plant cells are well known in the art. Suitablemethods include any method by which DNA can be introduced into a cell,such as by Agrobacterium mediated transformation, via bombardment by DNAcoated particles, direct delivery of DNA such as, for example, byPEG-mediated transformation of protoplasts, bydesiccation/inhibition-mediated DNA uptake, by electroporation, or byagitation with silicon carbide fibers.

In certain embodiments, the methods further comprise regenerating atransgenic plant from the transgenic plant cell. This step is performedprior to recovering a recombinant protein expressed by the transgenicplant. In certain aspects, the methods may comprise a recovery step thatfurther comprises obtaining an extract of the plant cell. In certainaspects, the methods may comprise harvesting material from at least aportion of the transgenic plant. In certain embodiments, at least aportion of the material harvested from the transgenic plant is edible.

Preferably, the methods directed to transforming a plant cell maycomprise the use of an Agrobacterium system.

In certain embodiments of the invention, a recombinant protein isexpressed in a transgenic plant, at least a portion of which plant isedible. In certain aspects, the recombinant protein may be administeredto a subject by oral administration, wherein a portion of the plantexpressing the recombinant protein is ingested by the subject, such thatan effective amount of the recombinant protein is delivered to thesubject.

The invention relates, in certain aspects to a method for constructing atransgenic plant cell comprising the steps of: constructing a plasmidvector or a DNA fragment by operably linking a DNA molecule, the DNAmolecule comprising a sequence encoding a protein, to a promoter capableof directing the synthesis of the protein in the plant; and transforminga plant cell with the plasmid vector or DNA fragment; preferably, theplant is a member of the genus Eriodictyon. In certain embodiments, theplant is preferably E. californicum, E. trichocalyx, or E.sessilifolium.

Preferably, the method of transforming the plant cell comprises the useof an Agrobacterium system.

In certain embodiments, the method may further comprise regeneratingtransgenic plants from the transgenic plant cell.

In certain aspects, the present invention relates to several factorsthat influence efficiency of Agrobacterium-mediated transformation. Forexample, the invention relates to a method for transforming a planttissue comprising the steps of: inoculating a transformable plant tissuewith an Agrobacterium suspension diluted to about OD₆₀₀ 0.005 to 0.5,the Agrobacterium containing at least one genetic component encoding adesired protein capable of being transferred to the transformable planttissue and of directing the expression of the desired protein in theplant tissue; co-cultivating the plant tissue with the Agrobacterium;transferring the plant tissue to recovery media containing an antibioticfor eliminating the Agrobacterium; and selecting transformed planttissue. In certain embodiments, the plant tissue is from a plant speciesof the genus Eriodictyon, preferably Eriodictyon californicum,Eriodictyon trichocalyx and Eriodictyon sessilifolium.

As used herein, “OD₆₀₀” means the optical density measure at awavelength of 600 nanometers. In a preferred embodiment, the inoculatingstep is performed with an Agrobacterium suspension diluted to aboutOD₆₀₀ 0.01 to 0.05, more preferably about OD₆₀₀ 0.03.

In certain embodiments, the length or temperature of the steps in thetransforming method must be precisely monitored to achieve transformedplants. For example, the inoculating step may be performed for about 1to 30 minutes, preferably for about 5 to 15 minutes, and more preferablyfor about 10 minutes. Likewise, the co-cultivating step may be performedfor about 1 to 4 days, preferably for about 2 days, at about 20 to 25°C. Finally, the plant tissue may remain in the recovery media for about5 to 20 days, more preferably for about 10 days.

In other embodiments, the method for transforming a plant tissue furthercomprises transferring the plant tissue to media containing successivelyhigher concentrations of a selective agent. Typical selective agentsinclude but are not limited to antibiotics such as kanamycin, geneticin,paromomycin or other chemicals such as glyphosate. In one embodiment,the selective agent is kanamycin and the genetic component of theAgrobacterim is capable of conferring kanamycin resistance to the planttissue.

The present invention, in certain aspects, also relates to methods forproducing a recombinant protein in a transgenic plant of the genusEriodictyon comprising the steps of: providing a transgenic plant thathas been regenerated from a transformed plant cell or tissue of thegenus Eriodictyon and that expresses a recombinant protein; andrecovering the protein expressed in the transgenic plant. Preferably,the plant is from the species of E. californicum, E. trichocalyx, or E.sessilifolium. In certain embodiments, the recovery step furthercomprises obtaining an extract of the transgenic plant or harvestingmaterial from at least a portion of the transgenic plant such as anedible portion of the plant.

The method of producing a recombinant protein in a transgenic plant, incertain embodiments may include using an Agrobacterium system, asdescribed herein, to transform the plant cell or tissue.

Preferably, the recombinant proteins produced by this method aresuitable for use as pharmaceuticals, including, but not limited to,antigens; microbicides; antibodies; hormones; enzymes; blood components,including, but not limited to, coagulation factors; interferons, andanticoagulants.

In certain embodiments, the method involves producing an antigenprotein, preferably a viral protein, more preferably an avian influenzaHA1 antigen.

In other embodiments, the method involves producing a microbicide,preferably an antiretroviral microbicide, more preferably an HIV entryinhibitor, even more preferably griffithsin.

The present invention, in certain aspects, also provides methods ofdelivering a recombinant protein to a subject comprising providingharvested material from a transgenic plant of the genus Eriodictyon thatexpresses a recombinant protein; and administering the harvestedmaterial to the subject in an amount necessary to deliver an effectiveamount of the recombinant protein.

In certain embodiments, the method of administration comprises deliveryto the subject of an edible portion of the transgenic plant expressing arecombinant protein. In other embodiments, the method of administrationmay comprise mucosal delivery to the subject of recombinant protein ormaterial containing such protein harvested from a transgenic plantproduced according to an aspect of the invention.

As used herein, the term “subject” is used to mean an animal, preferablya mammal, including a human. The terms “patient” and “subject” may beused interchangeably. Thus, certain embodiments of the invention aredirected to appropriate dosage forms useful in the administration ofactive pharmaceutical ingredients to a subject.

As used herein, “mucosal surface” includes, without limitation, nasal,oral, lingual, sub-lingual, buccal, gingival, palatal, vaginal, ocular,auditory, pulmonary tract, urethral, and rectal surfaces. Certainembodiments of the invention relate to compositions and methods foradministration of pharmaceutical agents to a mucosal surface in asubject.

A composition comprising a recombinant protein produced according to anaspect of the invention may be applied to any mucosal surface as deemedappropriate for the delivery thereof, including vaginal, rectal, andocular surfaces. In certain preferred embodiments, the method ofdelivery the mucosal surface is to an intranasal or oral surface of thesubject. Preferably, the oral surface may include, but is not limitedto, lingual, sub-lingual, buccal, gingival, and palatal surfaces.

In certain preferred embodiments, the recombinant protein comprises anantigen, preferably a viral antigen, more preferably an avian influenzaHA1 antigen. In this embodiment, the harvested material which containsthe expressed antigen is administered in an amount sufficient to inducean immune response in the subject. The immune response may include theinduction of cytotoxic T lymphocytes or the generation of antibodies.Preferably, the delivery of the antigen to the subject will produce asufficient immune response to confer resistance to infection upon thesubject. In any event, the method may be used to generate antibodies tothe antigen which may be used to aid in the purification of the antigen.Additionally, any generated antibodies may be useful in the detection ofthe virus from which the antigen is derived.

In other embodiments, the recombinant protein is a microbicide,preferably an antiretroviral microbicide, more preferably an HIV entryinhibitor, even more preferably griffithsin. In this embodiment, theharvested material which contains the expressed microbicide isadministered in an amount sufficient to provide a prophylactic effect.For example, harvested material from Yerba Santa expressing themicrobicide griffithsin may be administered to the mucosal surfaces(such as the vaginal or rectal mucosa) of subjects to protect thesesurfaces from HIV transmission.

The present invention, in certain aspects, also provides methods ofpropagating a plant in vitro, wherein the plant is preferably a memberof the genus Eriodictyon, the method comprising the steps of: excising astem segment, preferably a segment having a node; and incubating thesegment in a growth medium, preferably MS medium, the medium preferablycomprising a cytokinin, more preferably zeatin; whereby the segmentproduces a shoot. Preferably, the plant so propagated is E.californicum, E. trichocalyx, or E. sessilifolium.

As used herein, “cytokinins” refer to a class of plant hormones thatpromote cell division. Examples of cytokinins include the adenine-typecytokinins, kinetin, zeatin, benzylaminopurine (BAP); and thephenylurea-type cytokinins, diphenylurea and thidiazuron.

In certain embodiments, the methods may further comprise the steps of:excising a shoot; and, preferably, incubating the excised shoot inmedium, preferably MS medium, the medium preferably comprising an auxin,more preferably indole-3-butyric acid; whereby the shoot produces aroot.

As used herein, “auxins” refer to a class of plant hormones that controlcell expansion. Examples of auxins include the naturally occurringauxins, 4-chloro-indoleacetic acid, phenylacetic acid (PAA), andindole-3-butyric acid (IBA), indole-3-acetic acid (IAA); and thesynthetic auxins, naphthaleneacetic acid (NAA) and2,4-dichlorophenoxyacetic acid (2,4-D).

The methods of the invention may further comprise the steps of:incubating the shoot, preferably for at least three weeks, in culturemedium, whereby the shoot produces a leaf. The methods may furthercomprise the steps of: cutting a segment from the leaf; placing thesegment in a culture medium; and incubating the segment in the dark,whereby the segment develops callus tissue. Preferably, the medium is MSmedium. Preferably, the medium at least one of BAP, NAA and 2,4-D. Morepreferably, the medium comprises each of BAP, NAA, and 2,4-D.

In certain aspects, the invention relates to methods for rapid masspropagation of three Yerba Santa species in vitro, root induction,induction of cell suspensions, regeneration, transformation and transferof plants to greenhouse conditions. Additionally, methods for theproduction of callus tissues for all three species and for theestablishment of tissue culture, including fast growing cellsuspensions, are provided. Rapid and high frequency regeneration fromleaf explants is efficient for these three Yerba Santa species. Incertain embodiments, an efficient transformation protocol is providedand used for the production of an avian flu antigen or griffithsin inYerba Santa leaf tissues. Overall these results provide additionalopportunities to utilize and expand on the beneficial properties of thisunique medicinal herb.

In certain aspects, the invention relates to methods of producing a cellsuspension culture of a plant, preferably a plant that is a member ofthe genus Eriodictyon, the method comprising the steps of: excising aportion of the callus tissue produced according to certain aspects ofthe invention; placing the callus tissue in a liquid medium, preferablyMS medium, the medium preferably comprising 2,4-D; and incubating thesuspension in the dark with agitation.

In certain embodiments, the invention relates to protocols for in vitropropagation, callus induction, shoot regeneration, establishment of arapid growing cell suspension culture, and propagation in greenhouseconditions for Yerba Santa. Rapid and high frequency regeneration fromleaf explants (via shoot organogenesis) was efficient for all testedYerba Santa species. An Agrobacterium —mediated transformation protocolwas established. In certain aspects, transformation conditions areprovided for successful production of transgenic Yerba Santa, including,for example, the use of a precultivation period, low concentration ofAgrobacterium inoculums, and a multi-step selection procedure.

Yerba Santa species demonstrated several challenges in tissue cultureand transformation experiments including browning of explants ondifferent media, difficulties with root induction, high levels ofnecrosis after co-cultivation with Agrobacterium and selectionprocedures. Some of these difficulties are often encountered for othermedicinal plants. In certain embodiments of the invention, efficienttissue culture and transformation protocols for Yerba Santa are providedwhich may potentially find use with other recalcitrant medicinal plants.

Certain aspects of the invention relate to rapid mass propagation ofthree species of Yerba Santa. In vitro propagation may permit theproduction of pathogen-free material. Propagation from nodal stemsegments may yield plants that are genetically identical with the donorplants. In certain aspects, techniques described herein may providemethods for rapid propagation on a commercial scale of these medicinallyimportant plant species.

According to particular aspects of the invention, Yerba Santa cellsuspensions are provided, which are demonstrated to be very fast growingand not to contain large aggregates. This overcomes some difficultiesthat may be encountered in developing and maintaining an efficient cellsuspension from different plant species; including a slow rate of cellgrowth and the formation of large clumps during the culture period.

These plant-cell—suspension cultures may be used for the production ofrecombinant proteins, including, but not limited to, vaccines (Fischeret al., (1999) Journal of Immunol Methods 226:1-10); in certainembodiments, this production may be performed under controlled certifiedconditions. The Yerba Santa suspensions described herein have very goodgrowth rates, comparable to other fast-growing suspensions described inthe relevant literature.

The following examples are provided for the purpose of furtherillustrating the present invention but are by no means intended to limitthe same.

EXAMPLES Materials and Methods

Plant Material and Propagation In Vitro

Plant material of three Yerba Santa species (Eriodictyon californicum,Eriodictyon sessilifolium, and Eriodictyon trichocalyx) are obtainedfrom Rancho Santa Ana Botanic Garden (Claremont, Calif.) and Las PilitasNursery (Santa Margarita, Calif.). Segments of stem are excised fromYerba Santa plants, washed thoroughly under running tap water, and thenare dipped in 70% ethanol for 1 min, followed by a 25 min soak in a 1.2%solution of commercial sodium hypochlorite. After rinsing 3 times withsterile distilled water, segments with 1 or 2 nodes are transferred toPhytatrays containing MSP media. Phytatrays are sealed with Parafilm andincubated in a growth chamber at 24° C. at 16 h-light/8 h-darkphotoperiods with light intensity of 40 uE/m2/S1. In vitro cultures aremaintained by transferring 1-cm-long shoot segments at 5-6 weekintervals onto fresh medium. For root induction 3-4 cm shoots are placedin root induction media MSRI (Table 1). Rooted plants are transferred topods containing soil Metromix and sand (3:1).

TABLE 1 Media for tissue culture and transformation experiments NameMedia composition MS0 Basic MS basal medium with 3% sucrose, 0.8% agarMSP MS0 with 1 mg/l zeatin MSC-1 MS0 with 0.3 mg/l BAP, 0.5 mg/l NAA, 1mg/l 2,4-D MSC-2 MS0 with 1 mg/l 2,4-D MSS MS with 3% sucrose, 0.5 mg/l2,4-D (liquid) MSR MS with 3% sucrose, 1 mg/l zeatin, 0.1 mg/l NAA, 5mg/l silver nitrate 0.9% agar MSRI MS0 with 1 mg/l lBA MST-1 MS0 with100 μM acetosyringone MST-2 MSR with 300 mg/l timentin MST-3 MSR with 50mg/l kanamycin, 300 mg/l timentin MST-4 MSR with 70 mg/l kanamycin, 250mg/l timentin MST-5 MSR with 100 mg/l kanamycin, 200 mg/l timentin MST-6MS0 with 1 mg/l lBA, 150 mg/l timentin

Callus Initiation, Cell Suspensions

Leaf segments (0.5-0.7 cm) are cut from the 3-4 week-old in vitropropagating shoots from three Yerba Santa species and placed in Petridishes (100×15 mm) on MSC-1 medium for induction of callus. Plates areincubated in the darkness at 24° C. for 4-6 weeks. Well developed calliare selected and transferred to MSC-2 medium. Callus tissue ismaintained on MSC-2 medium at 3- to 4-week intervals. All the plateswith callus cultures are incubated in the dark at 24-25° C. Friablecallus is used for initiation of cell suspensions. Approximately 1 gfresh weight of callus tissue are transferred into 50 ml of medium MSSmedium in sterile 250 ml conical flasks. Cell cultures are grown on arotary shaker at 130 rpm in the dark at 25° C. In order to maintainsuspension culture a portion (2-3 ml) of liquid suspension cells aretransferred to fresh MSS medium at 10-14 day-intervals.

Generation of Transgenic Plants

Leaf segments (0.5-0.7 cm) are cut from the 3-4 week-old in vitropropagating shoots of three Yerba Santa species and placed in Petridishes (100×15 mm) on MSR medium. Ten to twelve explants per Petri dishare cultivated for 6 weeks and tested for shoot regeneration efficiency.Agrobacterium tumefaciens strain LBA 4404 is grown overnight in LBmedium supplemented with appropriate antibiotics at 28° C. Binary vectorpBIN-Plus (ImpactVector, Wageningen, the Netherlands) harboring anexpression cassette of avian flu HA1 antigen fused with Fc and driven bythe Rubisco promoter is used. The expression cassette contains the c-Mycand His6 tags at the C-terminus. The vector also contains the npt IIgene for kanamycin selection of transgenic plants. Leaf segments areinoculated with Agrobacterium suspension (OD₆₀₀ 0.5, 0.3, 0.1 or 0.03)for 10 min. After blotting dry with sterile filter paper, explants aretransferred to MST-1 co-cultivation medium supplemented withacetosyringone (Table 1) and incubated in the dark for 2 or 3 days at24° C. To determine the effect of preculture on transformationefficiency, explants are cultured for 2, 3 or 4 days on MSC-1 mediumbefore inoculation with Agrobacterium. After co-cultivation, explantsare transferred to MST-2 medium without selection for 7, 10 and 14 daysand then transferred to MST-3 regeneration selection medium with 50 mg/lkanamycin. After 3 weeks, regenerated green shoots are transferred tosecond selection medium MST-4 with increased concentration of kanamycin(70 mg/l), and after an additional 3 weeks explants are transferred tothird selection medium MST-5 with high kanamycin concentration (100mg/l). Putative transgenic shoots are excised and transferred to rootingmedium (MSRI supplemented with 150 mg/l timentin). Plantlets with rootsare transferred to pots containing a mixture of soil and sand.

Example 1 In Vitro Propagation and Root Induction

For establishment of in vitro cultures stem segments with nodes of threeYerba Santa species (Eriodictyon californicum, Eriodictyonsessilifolium, and Eriodictyon trichocalyx) are used as primaryexplants. They are washed, surface sterilized and placed on MS medium(Murashige T& Skoog F.(1962) Physiol Plant 15:473-497) supplemented withdifferent cytokinins: N⁶-benzylaminopurine (BAP), zeatin, and kinetin.All three Yerba Santa species propagate in vitro most efficiently onbasal MS medium supplemented with 1 mg/l zeatin. During 6 weeks asignificant number of shoots are produced for all three species.Comparison of the shoot propagation capacity of three species of YerbaSanta demonstrates the highest efficiency in E. trichocalyx (FIG. 1, Aand B), about 8-10 shoots are produced from each explant in 4-5 weeks. Asingle explant of this species can produce thousands of shoots in 3-4months.

Individual shoots are excised and transferred to hormone-free medium forrooting. Yerba Santa species demonstrate different root formationcapacities. E. sessilifolium start to form roots after 7-10 days onmedia without hormones (FIG. 1C), whereas E. californicum and E.trichocalyx show significant difficulties with root formation. Thereforemedia with different auxins are tested for stimulation of rootformation: naphthalene acetic acid (NAA), indole-3-acetic acid (IAA),and indole-3-butyric acid (IBA). The best root development for bothcultivars is recorded on medium supplemented with 1 mg/l IBA (FIG. 1, Dand E), however some of the shoots are not able to produce roots. Aftertesting shoots of different size and development, it appears that large(3-4 cm), healthy and well developed shoots of E. californicum and E.trichocalyx are capable of producing roots. Rooted plants (FIG. 1F) aretransferred to pods with mixture of soil (Metromix) and sand (3:1)(FIGS. 1G and 1H). Morphology of the micropropagated plants areidentical to that of normal propagated plants.

Example 2 Establishment of Cell Suspensions

To obtain a rapidly growing cell suspension culture, three Yerba Santaspecies are screened for callus induction and cultivation. Callustissues are initiated for all three Yerba Santa species from leafexplants on callus induction media MSC-1 after 4-6 weeks of incubationin darkness. For future propagation, callus tissues are transferred toMSC-2 medium. After 6-8 weeks of cultivation on MSC-2 medium, the callusof E. trichocalyx showed the best growth capacities (FIG. 2A) E.californicum also demonstrated good growth capacity, however some partof the callus tissues showed browning (FIG. 2B).

Cell suspensions of Yerba Santa species are established from callustissues in liquid media MSS. E. trichocalyx show the best results,including fast growth rates of cells, no browning of cells, and mildcell aggregations (FIGS. 2C and 2D). The E. trichocalyx cell suspensionis very fast growing: about a 10-12-fold increase in cell volume isachieved in 5-7 days. (FIG. 3). These results are comparable with mostfast growing cell suspension of other plant species (Fischer R &Schillberg S (eds) (2004) Molecular Farming. Plant-made Pharmaceuticalsand Technical Proteins Wiley-VCH Verlag GmbH & Co. Weinbeim) and canprovide a very efficient system for the production of vaccines and otherrecombinant proteins.

Example 3 Transformation of Yerba Santa

As a first step in the development of an efficient transformationsystem, the development of an efficient regeneration system isinitiated. Preliminary experiments using different media compositionsand types of explants indicate that shoots from several Yerba Santaspecies regenerate most efficiently on MSR medium (Table 1) and thatleaf segments have the best regeneration potential. During 5-6 weeksleaf explants produce multiple shoots on MSR medium through directregeneration without callus formation. Comparison of the regenerationcapacity of three species (E. californicum, E. sessilifolium, and E.trichocalyx) reveal the highest regeneration efficiency in E.trichocalyx leaf segments reaching 75%-82% (FIG. 4; FIGS. 5A and 5B).Based on the results of regeneration experiments, leaf segments of E.trichocalyx are chosen as explants for transformation experiments.

Preliminary transformation experiments reveal several challengesassociated with inoculation and selection for Yerba Santa species. Inthe first series of experiments, the efficiency of the inoculationprocedure is tested. The exposure of leaf explants to Agrobacteriumculture at OD₆₀₀ 0.5 cause severe necrosis in most of the treated YerbaSanta explants. Pre-cultivation of E. trichocalyx leaf explants istested for 2, 3 and 5 days before inoculation with Agrobacterium OD₆₀₀0.5. However, this approach does not appear to decrease the number ofbrowning tissues after inoculation and co-cultivation procedures. In aneffort to reduce necrosis of explants in response to Agrobacteria, theAgrobacteria suspension is diluted to OD₆₀₀ 0.3, 0.1, and then finallyto 0.03. Inoculation of leaf segments with a suspension diluted to 0.03significantly decreases the level of necrosis. In the same set ofexperiments, it is demonstrated that 2 days of co-cultivationsignificantly decreased necrosis, as compared to 3 days. The addition ofpolyvinylpyrrolidone (250 mg/l) and increasing the agar concentration inthe media also prove to be beneficial.

Several selection schemas are tested. No transgenic plants are recoveredwhen selection is started immediately after co-cultivation. Therefore,the effect of a delay period is tested, during which explants are keptin MST-2 non-selection medium supplemented with timentin forAgrobacterium elimination for 7, 10 or 14 days. Transgenic shootregeneration from explants is highest when leaf explants are left onMST-2 medium for 10 days, then explants are transferred to selectionmedium. Among different selection systems tested the best results areobtained with a 3-step selection procedure with a gradually increasingconcentration of kanamycin in the selection regeneration media. Use of arelatively low concentration of kanamycin (50 mg/l) in the firstselection media reveals good survival of tissues and a large percent ofescapes. After 3 weeks, the explants are transferred to a secondselection medium containing 70 mg/l kanamycin and finally to a selectionmedium with 100 mg/l kanamycin (FIG. 5C). When shoots reach 3-4 cm inlength (FIG. 5D), they are transferred to root induction media MSRI withaddition of 150 mg/l timentin (FIG. 6A). Rooted plants are transferredto pods with mixture of soil and sand (FIG. 6B).

Example 4 Expression of Avian Influenza Antigen

Leaf tissues of transgenic plants are tested for level of expression ofavian influenza antigen. Western blot analysis with C-myc antibodiesreveals a protein band of the expected molecular size in the leaf tissueof transgenic plants (FIG. 7). The morphology of the transgenic plantsis identical to that of non-transgenic plants (FIG. 6B and FIG. 1H).

Example 5 Immunological Assessment of Plant-Expressed Avian InfluenzaAntigen in Mice

Groups of 6- to 8-week-old female BALB/c mice (five mice per group) areused in all experiments. The experiment is performed using Eriodictyoncalifornicum, Eriodictyon sessilifolium, or Eriodictyon trichocalyxplants that have been transformed to express HA1 antigen and wild type(WT) plants.

For oral immunization experiments, groups of 6- to 8-week-old femaleBALB/c mice (five mice per group) are used. The experiment are performedusing Yerba Santa plants that express HA1 antigen and wild type (WT)plants. Each mouse is fed with 2-3 g of fresh leaf tissue over a periodof 6-8 h. Control mice receive wild type plant material. Mice areimmunized 3 times at 2-week intervals.

For intranasal immunization experiments, groups of 6- to 8-week-oldBALB/c mice (five per group) are used. 2 μg of Yerba Santa-derivedantigen is administered in 10 μl of saline into both nostrils (5 μl ineach). In some groups, plant material is supplemented with 1 μg of CT(cholera toxin) as an adjuvant. Control mice receive wild type plantmaterial. Mice are immunized 3 times at 2-week intervals.

Blood and fecal matter is collected 10 days after each immunization.Protein from fecal pellets will be extracted in PBS (10 vol/wt)supplemented with 1% BSA and protease inhibitors. Mice are killed 10days after the last immunization and bled by cardiac puncture. Sera andfecal pellets are analyzed for the presence of antigen (HA1) specificantibodies by Western blot analysis and ELISA.

Solid-phase ELISA is carried out as described in Hooper et al. (2001) J.Immunol. 167, 3470-3477 MaxiSorp 96-well plates (Nalge Nunc) are coatedovernight at 4° C. with the HA1 at a concentration of 1 μg/ml in PBS.Extract are diluted initially 1:10 in PBS and diluted serially 1:2 inthe same buffer incubation for 1 hour at 37° C. Antigen-specificantibodies are detected by using the following antibodies: rabbitanti-mouse IgG (total) and anti-mouse IgG1 (both from BD BiosciencesPharmingen), anti-mouse IgG2a, IgG2b, IgG3 and IgA (all from OrganonTeknika), and anti-mouse IgE (eBioscience, San Diego) HRP-conjugated(diluted 1:2000 in PBST) for 1 h at 37 C. Between each step, wells arewashed four times with PBST. Finally, plates are developed in a solutionof OPD peroxidase substrate (Sigma Chemical). Absorbance at 490 nm isdetermined using a microplate reader.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and scope of the invention, and all such variations are intendedto be included within the scope of the following claims.

In addition, where features or aspects of the invention are described interms of Markush group or other grouping of alternatives, those skilledin the art will recognized that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Unless indicated to the contrary, all numerical ranges described hereininclude all combinations and subcombinations of ranges and specificintegers encompassed therein. Such ranges are also within the scope ofthe described invention.

The disclosures of each patent, patent application and publication citedor described in this document are hereby incorporated herein byreference, in their entirety.

1. A transgenic plant of the genus Eriodictyon.
 2. The transgenic plantof claim 1, wherein the plant is selected from the group consisting ofE. californicum, E. trichocalyx and E. sessilifolium.
 3. The transgenicplant of claim 1, which expresses a recombinant protein.
 4. Thetransgenic plant of claim 3 wherein the recombinant protein comprises anavian influenza HA1 antigen.
 5. A method for transforming tissue from aplant of the genus Eriodictyon comprising the steps of: inoculating leaftissue from an Eriodictyon plant with an Agrobacterium suspensiondiluted to about OD₆₀₀ 0.005 to 0.5, the Agrobacterium containing atleast one genetic component encoding a desired protein capable of beingtransferred to the transformable plant tissue and of directing theexpression of the desired protein in the plant tissue; co-cultivatingthe plant tissue with the Agrobacterium; transferring the plant tissueto recovery media containing an antibiotic for eliminating theAgrobacterium; and selecting transformed plant tissue.
 6. The method ofclaim 5 wherein the plant species is selected from the group consistingof Eriodictyon californicum, Eriodictyon trichocalyx and Eriodictyonsessilifolium.
 7. The method of claim 5 wherein the Agrobacteriumsuspension is diluted to about OD₆₀₀ 0.03.
 8. The method of claim 5wherein the co-cultivating of the plant tissue with the Agrobacteriumsuspension is for about 1 to 4 days at about 20 to 25° C.
 9. The methodof claim 8 wherein the co-cultivating of the plant tissue with theAgrobacterium suspension is for about 2 days.
 10. A method for producinga recombinant protein in a transgenic plant of the genus Eriodictyoncomprising the steps of: providing a transgenic plant that has beenregenerated from a transformed plant cell or tissue of the genusEriodictyon and that expresses a recombinant protein; and recovering therecombinant protein expressed in the transgenic plant.
 11. The method ofclaim 10 wherein the recovery step further comprises obtaining anextract of the transgenic plant.
 12. The method of claim 10 wherein therecovery step further comprises harvesting material from at least aportion of the transgenic plant.
 13. The method of claim 12 wherein theat least a portion of the harvested material is edible.
 14. The methodof claim 10 wherein the plant cell or tissue is transformed using anAgrobacterium system.
 15. The method of claim 14 wherein the plant cellor tissue is transformed using the method of claim
 5. 16. The method ofclaim 10 wherein the recombinant protein is selected from the groupconsisting of antigens, microbicides, antibodies, hormones, enzymes,blood components, interferons, and anticoagulants.
 17. The method ofclaim 16 wherein the antigen comprises a viral protein.
 18. The methodof claim 17 wherein the protein comprises an avian influenza HA1antigen.
 19. The method of claim 16 wherein the microbicide comprises anantiretroviral.
 20. The method of claim 19 wherein the antiretroviralcomprises griffithsin.